Purα (also known as Pur-alpha, Pur-α, or Puralpha), Cyclin T1, and interferon regulatory factors (“IRFs”) 3 and 7 are all naturally occurring proteins that are involved in nucleic acid regulatory processes in healthy and unhealthy mammalian cells, including those of humans. They are also involved in the transmission and replication of viral pathogens, including HIV, polyomaviruses and species of bacteria, including those causing Lyme disease and syphilis, as well as protists including those causing malaria. Trans-activator of transcription (“Tat”) is a HIV gene which, through the involvement of partner proteins (such as Purα, cyclin T1, and IRF3 and 7), increases the level of transcription of HIV DNA. The interactions between Purα, Cyclin T1, and IRFs 3 and 7 with Tat cause HIV transcription and co-opting of cellular functions. There is a need for synthetic agents that will interfere with Tat and these protein binding partners in order to prevent or reduce the spread of HIV infection. In addition, Purα, Cyclin T1, and IRFs 3 and 7 are involved in transcription and cellular processes. For example, Purα is involved in regulating both cell growth and cell fate in cancer. Therefore, there is also a need to harness the beneficial properties of the Purα protein in order to provide therapies to treat or prevent cancers. The common denominator in Purα function, tying together its actions in all organisms, is its ability to bring proteins and nucleic acids together in such a way that these two classes of macromolecules can move relative to each other. A value of this invention is that it can inhibit pathological aspects of Purα function while allowing Purα functions necessary for healthy cell activities. The polypeptides of the invention are also valuable due to their ability to be delivered.
Purα
Purα is a sequence-specific single-stranded DNA-binding protein that functions in binding both DNA and RNA. It binds to purine-rich (“Pur”) elements found in the promoters of genes, and has a great affinity for DNA elements upstream of the c-MYC gene (Bergemann, A. D. and Johnson, E. M. (1992) The HeLa Pur factor binds single-stranded DNA at a specific element conserved in gene flanking regions and origins of DNA replication. Mol. Cell. Biol. 12, 1257-1265; Bergemann, A. D., Ma, Z.-W. and Johnson, E. M. (1992) Sequence of cDNA comprising the human pur gene and sequence-specific single-stranded-DNA-binding properties of the encoded protein. Mol. Cell. Biol. 12, 5673-5682; Johnson, E. M., Anticancer Res., 23:2093-2100 (2003)). Purα can act as either a transcriptional activator or a repressor. (D. Silva, N., A. Bharti, and C. S. Shelley, Blood, 100:3536-3544 (2002); Gallia, G. L., E. M. Johnson, and K. Khalili, 28:3197-3205 (2000)).
Purα is multifunctional in nature, as it is involved in DNA replication, RNA transcription, RNA transport, viral protein interactions, and regulation of viral replication at low concentrations. More specifically, Purα is involved in regulating both cell growth and cell fate. For example, Purα regulates progression of the cell cycle.
The cell cycle is a sequence of events, including interphase and the mitotic phase, from the time a eukaryotic cell divides to form two daughter cells to the time the daughter cells divide again. The cycle consists of four phases, gap 1 (“G1”), synthesis (“S”), gap 2 (“G2”), and mitosis (“M”). Interphase occurs from G1 phase through the G2 phase. During G1, the cell increases in size, and increases its supply of proteins and the number of many of its organelles (e.g., mitochondria and ribosomes). Following G1, S phase occurs. DNA synthesis (replication) occurs during S phase, and after DNA replication, single chromosomes are present as double chromosome, each consisting of two sister chromatids. The third subphase, G2, spans the time from the completion of DNA synthesis to the beginning of cell division. During this time, proteins that are essential to cell division are made by the cell. During the fourth subphase, M, cytokinesis and mitosis occur. Cytokinesis is the process by which the cytoplasm (cytokinesis) divides and is distributed to form two daughter cells. Mitosis is the process by which the nucleus and its contents, including the duplicated chromosomes, divide and are distributed to form two cells.
The introduction or expression of Purα during various phases of the cell cycle can be used to regulate cell growth. Purα interacts directly with retinoblastoma protein (“Rb”), cyclin-dependent kinase (“Cdk2”), and cell division cycle 6 (“Cdc6”) in a dose dependent manner to affect cell determination after oncogenic stress. For example, microinjection of Purα halts deregulated cell growth by arresting cell-cycle progression at the G1 or G2/M phases, depending upon the cell cycle phase during which Purα is injected. During S phase, cyclin A must bind with Cdk2 for the cell to progress normally through the S phase. Purα co-localizes with cyclin A/Cdk2 in S and G2 to interrupt this process. Specifically, Purα recruits Cdk2 to specific Purα binding sites. The interaction of Purα with Cdk2 stimulates histone phosphorylation and displaces the kinase inhibitor, p21, to affect chromatin structure. (Liu, H., S. M. Barr, C. Chu, D. S. Kohtz, Y. Kinoshita, and E. M. Johnson, Biochem. Biophys. Res. Commun., 328:851-7 (2005)). Purα further alters chromatin structure by binding to Pur elements to cause local unwinding that affects DNA structure upstream and downstream. Thus, Purα has the ability to regulate cell growth by altering chromatin structure.